Glutathione S-transferase-pi (GST-P) is detected in rat liver altered hyperplastic foci and hepatocellular carcinoma and is considered the most reliable biomarker in rat hepatocarcinogenesis. Ito and coworkers (Toxicologist, 12:205, 1992) have used an in vivo assay system for GST-P induction for identifying carcinogenicity of 198 chemicals and reported it to be highly effective for identifying hepatocarcinogens but not for nonhepatocarcinogens. The reason for the ineffectiveness of Ito's assay system to identify nonhepatocarcinogens is probably because most of the nonhepatocarcinogens are direct acting or being activated at the site of application. We have used a cultured rat liver cell (GP6 cell line) system to study induction of GST-P by hepatocarcinogens, nonhepatocarcinogens, and noncarcinogens. GP6 cells at 100,000 cells/dish in Richter's medium +10% fetal calf serum and gentamycin were exposed to dimethylsulfoxide (DMSO) at 0, 0.1, 0.2, or 0.4%, phenobarbital (PB) at 1.5 mM, diethylnitrosamine (DEN) at 16 ul/ml, or dimethylbenzanthracene (DMBA) at 0.2 ug/ml in the presence of rat liver S9. The cells were refed with fresh medium containing the chemical every 3-4 days and subcultured weekly. Cytosolic GST-P activity was assayed by spectrophotometry using ethracrynic acid as substrate. Compared with controls, GST-P activity increased significantly in GP6 cells exposed to PB, DEN, or DMBA for 3-5 weeks. DMSO at up to 0.4% had no effect on GST-P activity. The results showed that the nonhepatocarcinogen DMBA, along with the hepatocarcinogens, DEN and PB, were effective in elevating GST-P activity in GP6 cells whereas the noncarcinogen, DMSO, was ineffective. This in vitro system using GST-P as a marker may be useful for identifying carcinogens including nonhepatocarcinogens since direct acting carcinogens such as DMBA can be studied.